Diabetes mellitus results from an absolute or relative deficiency of the hormone insulin. Although absolute deficits in the numbers of insulin-producing pancreatic β cells are seen in Type 1 diabetes, Type 2 diabetes often is accompanied by relative deficits in pancreatic β-cell mass and insulin production in the setting of insulin resistance (Butler, A. E., et al., Diabetes 52:102-110 (2003); Gerich, J. E., Mayo Clin. Proc 78:447-456 (2003); Yoon, K. H., et al., J Clin Endocrinol Metab 88:2300-2308 (2003)). Monogenic heritable forms of insulin-deficient, early-onset diabetes are associated with mutations in genes encoding factors essential for the transcriptional regulation of insulin production in pancreatic β cells (Fajans, S. S., et al., N Engil J Med 345:971-980 (2001)). Five of the six genes linked to maturity-onset diabetes of the young (MODY), hnf-4α/MODYI, hnf-1α/MODY3. ipf-1(pdx-1)/ MODY4, hnf-1β/MODY5, and neuroDl/MODY6, are transcription factors known to regulate endocrine cell development and/or the glucose-responsive expression of the insulin gene in pancreatic β-cells. Thus transcriptional regulators of pancreatic β-cell mass and insulin production represent an important pool of candidate diabetes genes.
Glucose-responsive regulatory regions that are highly conserved in the insulin promoters of multiple species serve as convergence points for β-cell transcription factors of the homeodomain and basic helix-loop-helix (bHLH) classes including several of the designated MODY genes (Steiner, D. F., et al., Annu Rev Genet. 19:463-484 (1985); German, M. S., et al, Genes Dev 6:2165-2176 (1992)). The physiologic regulation of insulin gene expression is dependent on the precise assembly of transcription factors and coactivators within specific concentration ranges. Multiple protein-protein interactions generate functional regulatory complexes that couple with the coactivators Creb-binding protein (CBP) or p300 in the transcriptional activation of the insulin gene (Ohneda, K., et al., Mol Cell Biol 20:900-911 (2000); Asahara, H., et al., Mol Cell Biol 19:8219-8225 (1999); Qiu, Y., et al., Mol Cell Biol 22:412-420 (2002); Stanojevic, V., et al., Endocrinology p. 300 145:918-928 (June 2004).
Bridge-1. By screening for novel interaction partners for the bHLH transcription factor E12 in a clonal β-cell line the present inventors previously identified Bridge-1 as a PDZ-domain coactivator of the insulin gene (Thomas M. K., et al., Mol Cell Biol 19:8492-8404 (1999)). Bridge-1 contains a highly conserved PDZ protein-protein interaction domain and is part of the large family of PDZ-domain proteins that facilitate the assembly of supramolecular protein complexes on PDZ-based scaffolds to transduce localized intracellular signals (Sheng, M., and Sala, C., Annu Rev Neurosci 24:1-29 (2001)). Rat Bridge-1 is a highly conserved, widely expressed protein with substantial homology to the human proteasomal modulator protein PSMD9 (Thomas, M. K., et al., Mol Cell Biol 19:8492-8404 (1999); Watanabe, T. K., et al., Genomics 50:241-250 (1998)). In the adult pancreas Bridge-1 is expressed predominantly in the insulin-expressing pancreatic βcells of the endocrine compartment. Bridge-1 interacts with E12 and E47 to coactivate glucose-responsive enhancers within the insulin promoter, and Bridge-1 antisense constructs substantially reduce insulin promoter activation in insulin-producing cells in vitro (Thomas, M. K., et al., Mol Cell Biol 19:8492-8404 (1999)). Therefore, the present inventors hypothesized that endogenous Bridge-1 signaling regulates insulin production in pancreatic β cells.
P300. Transcriptional coregulators provide important regulatory flexibility in the cellular responsiveness to hormones and extracellular signals. The physiologic importance of coregulator function is highlighted by the association of coactivator dysfunction with multiple human disease states including neurodegeneration, malignancies and metabolic disorders. The nuclear receptor coactivator p300 functions to assemble multimolecular transcriptional regulatory protein complexes through interactions with a large repertoire of transcription factors and components of basal transcription machinery (Vo, N. and Goodman, R. H., J. Biol. Chem. 276:13505-13508 (2001); Chan, H. M. and La Thangue, N. B., J. Cell. Sci. 114:2363-2373 (2001)). The intrinsic acetyltransferase activity of p300 augments the activation of gene transcription through acetylation of histones and transcription factors (Vo, N. and Goodman, R. H., supra). Mutations in the human p300 gene, like those in the related Creb-binding protein (CBP) gene, result in heritable tumors in the Rubinstein-Taybi syndrome (Roelfsema, J. H., et al., Am. J. Hum. Genet. 76:572-580 (2005); Giles, R. H., et al., Trends Genet. 14:178-183 (1998)). Somatic mutations in p300 also occur in sporadic tumors, consistent with the demonstrated function of this coactivator as a tumor suppressor in mouse models (Chan, H. M. and La Thangue, N. B., supra; Iyer, N. G., et al., Oncogene 23:4225-4231 (2004)). Altered levels of expression of p300 modify embryonic development, cellular functions, and responsiveness to extracellular signals (Yao, T. P., et al., Cell 93:361-372 (1998)). For example, in the case of Paget's disease, the hyperresponsiveness of osteoclast precursors to vitamin D is associated with increased expression levels of coactivators including p300 (Kurihara, N., et al., J. Ster. Biochem. Molec. Biol. 89-90:321-325 (2004)). In a mouse model of Huntington's disease, progressive deficits in insulin production are correlated with decreased expression levels of p300 and other transcription factors in insulin-expressing cells (Andreassen, O. A., et al., Neurobiol. Dis. 11:410-424 (2002)).
In early-onset autosomal-dominant heritable forms of diabetes known as maturity-onset diabetes of the young (MODY), abnormal interactions between mutant MODY transcription factors and p300 may contribute to deficits in insulin production and the pathogenesis of disease. p300 recruitment to the proximal insulin promoter in insulin-producing pancreatic beta cells is associated with hyperacetylation and transcriptional activation (Chakrabarti, S. K., et al., J. Biol. Chem. 278:23617-23623 (2003)), and p300 interactions with the transcriptional regulators PDX-1, NeuroD1, and E47 enhance insulin gene transcription (Qiu, Y., et al., Molec. Cell. Biol. 18:2957-2964 (1998)); Sharna, A., et al., Molec. Cell. Biol. 19:704-713 (1999); Qiu, Y., et al., Molec. Cell. Biol. 22:412-420 (2002); Mosley, A. L., et al., Molec. Endocrinol. 18:2279-2290 (2004); Stanojevic, V., et al., Endocrinol. 145:2918-2928 (2004)). Five direct or indirect transcriptional regulators of insulin gene transcription are encoded by genes associated with MODY (Fajans, S. S., et al., New Engl. J. Med. 345:971-980 (2001)). MODY1 mutations R154X and E276Q in the HNF-4α gene interfere with p300 recruitment and transcriptional activation (Eeckhoute, J., et al., Molec. Endocrinol. 15:1200-1210 (2001)), the MODY4 mutation P63fsdelC results in a truncated cytoplasmic PDX-1 protein with the capacity to sequester p300 (Stanojevic, V., et al., supra), and the MODY6 mutation 206+C disrupts NeuroD1 binding to p300 (Malecki, M. T., et al., Nat. Genet. 23:323-328 (1999)).
PDX-1. The homeodomain transcription factor pancreas duodenum homeobox-1 (PDX-1) is an important regulator of both the embryonic development of the pancreas as well as the function and mass of insulin-expressing pancreatic beta cells (Thomas et al., 2004). PDX-1 has also been referred to as IPF-1, STF-1, IUF-1, GSF, and IDX-1. Homozygous or compound heterozygous disruption of pdx-1 gene expression in mice or in humans results in a common phenotype of pancreatic agenesis (Jonsson et al., 1994; Offield et al., 1996; Schwitzgebel et al., 2003; Stoffers et al., 1997a). Partial reductions of PDX-1 expression levels in genetically-modified mouse models disrupt glucose homeostasis by reducing insulin expression, insulin secretion, and pancreatic beta-cell mass, in part via accelerated apoptosis of insulin-producing pancreatic beta cells (Ahlgren et al., 1998; Brissova et al., 2002; Dutta et al., 1998; Holland et al., 2002; Johnson et al., 2003; Thomas et al., 2001).
In humans, pdx-1 (ipf-1, insulin promoter factor-1) is a diabetes gene. Heterozygous inheritance of an inactivating mutation in pdx-1 results in autosomal-dominant maturity-onset diabetes of the young (MODY4) (Stoffers et al., 1997b) while heterozygous missense mutations in pdx-1 confer an increased risk of early- or late-onset type 2 diabetes in selected populations, often in combination with other diabetogenic genotypes (Cockburn et al., 2004; Elbein et al., 2004; Hani et al., 1999; Hansen et al., 2000; Macfarlane et al., 1999; Owen et al., 2004; Waeber et al., 2000; Weng et al., 2001; Weng et al., 2002).
PDX-1 is implicated in the transcriptional regulation of a large number of pancreatic islet genes, including insulin and somatostatin (Leonard et al., 1993; Miller et al., 1994; Ohlsson et al., 1993). Nutrient-dependent regulation of insulin gene expression is mediated in part through the regulation of PDX-1 nuclear translocation, DNA-binding and transcriptional activation by glucose (MacFarlane et al., 1994; MacFarlane et al., 1997; Melloul et al., 1993; Petersen et al., 1998; Rafiq et al., 1998; Shushan et al., 1999). PDX-1 participates in the synergistic activation of glucose-responsive enhancers with basic helix-loop-helix transcription factors, such as E12 and E47 (Massari et al., 2000), via its amino-terminal domain (German et al., 1992; Johnson et al., 1997; Peers et al., 1994).
The regulation of PDX-1 target genes also is governed by the interactions of PDX-1 proteins with other transcription factors and coactivators. Nuclear translocation of PDX-1 is regulated by interaction of the PDX-1 homeodomain with the nuclear import receptor importin beta1 (Guillemain et al., 2004). PDX-1 cooperatively activates the somatostatin promoter in conjunction with PBX and the PBX regulating protein-1 (Prep1) (Goudet et al., 1999). Interactions between PBX and a conserved amino acid motif (FPWMK) within PDX-1 are necessary for the proliferation of differentiated pancreatic cells during embryonic pancreas development (Dutta et al., 2001). PDX-1 recruits E47, Beta2/NeuroD1, and the high-mobility group protein HMG(Y) to the insulin promoter (Ohneda et al., 2000). The amino-terminal transactivation domain of PDX-1 (Lu et al., 1996; Peers et al., 1994) interacts with the coactivators Creb-binding protein (CBP) and p300 (Asahara et al., 1999; Qiu et al., 2002; Stanojevic et al., 2004). In contrast, the carboxy-terminal domain of PDX-1 serves as an interaction domain for transcriptional repressors, including phosphorylated carboxy-terminal domain interacting factor 1 (PCIF1) and the histone deacetylases HDAC1 and 2 (Liu et al., 2004; Mosley et al., 2004).